Arenaviruses merit significant interest both as tractable experimental model systems to study acute and persistent viral infections and as clinically important human pathogens. Several arenaviruses cause hemorrhagic fever (HF) disease in humans, whereas the prototypic Arenavirus LCMV is a superb workhorse for the investigation of basic concepts in the fields of viral immunology and pathogenesis. In addition, evidence indicates that LCMV is a neglected human pathogen of clinical significance. Our long- term objective is to obtain a detailed understanding of the Arenavirus molecular and cell biology. This knowledge will contribute to the elucidation of Arenavirus-host interactions and associated diseases, and facilitate the development of effective strategies to combat Arenavirus infections. To this end we have developed a reverse genetics system for LCMV that provides us with a novel and powerful approach for the investigation of Arenavirus biology. The focus of this proposal is to functionally characterize viral and cellular proteins, and their interactions, which mediate control of Arenavirus RNA synthesis, particle formation and viral budding. Our specific aims are: 1. To Assess the role of Z-NP In control of viral RNA synthesis. We will test the hypothesis that Z- NP interaction is responsible for the inhibitory activity of Z on viral RNA synthesis. We will use biochemical and genetic approaches to identify the regions of Z and NP required for Z-NP interaction, and use reverse genetic approaches to examine the functional consequences of disrupting Z-NP interaction. 2. Determine the role of Z-GP interaction in production of arenavirus infectious particles. We have demonstrated that production of infectious VLP requires Z and GP, and shown that Z and GP interact. We will define biochemically and functionally this Z-GP interaction. We will test the hypothesis that the correct Z-GPcx association is required for the production of infectious particles, and that this interaction is mediated by defined regions within Z and the GP-2 component of the GP complex (GPcx). 3. Determined the function of Z L domain motifs during the natural course of Arenavirus infection. We will use reverse genetics to rescue rLCMV carrying Z proteins with different types of L-domains. These rLCMV will be used in cell culture and mice models of infection to test the hypothesis that the type of L- domain motifs present in Z influences viral growth and virulence. 4. Identify and functionally characterize host proteins that influence Z-mediated budding. We will test the hypothesis that Z proteins containing different L-domains engage distinct subsets of Class E proteins of the MVB pathway, and that these differences influence virus-host interactions and associated diseases. We will use proteomic approaches to define the Z interactome. As a complementary approach we will use siRNA-based screenings to identify cellular factors that contribute to Z-mediated budding. We will use biochemical, genetics and functional assays to assess the relevance in Arenavirus biology of candidates initially identified by proteomic and genetic approaches. PUBLIC HEALTH RELEVANCE: [unreadable] Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and mounting evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is a neglected human pathogen of clinical significance. Moreover, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Therefore it is important to develop novel antiviral strategies to combat arenaviral infections. A detailed understanding of the arenavirus molecular will facilitate this task and cell biology, which is the long-term goal of the studies proposed in this application. [unreadable] [unreadable] [unreadable]